Glioblastoma is the most frequent primary brain tumor in adults and a highly lethal malignancy with a median survival of about 15 months. The aggressive invasion of the surrounding normal brain makes complete surgical resection impossible, increases the resistance to radiation and chemotherapy, and virtually assures tumor recurrence. Thus, there is a significant unmet clinical need to develop innovative approaches to target the invasive tumor cells for improved treatment of this disease. Expression of TROY, a member of the TNFR family, increases with increasing glial tumor grade and inversely correlates with patient survival. Increased expression of TROY stimulates glioblastoma cell invasion in vitro and in vivo and increases resistance to temozolomide and radiation treatment. Conversely, silencing TROY expression inhibits glioblastoma cell invasion, increases sensitivity to temozolomide, and prolongs survival in an intracranial xenograft model. Preliminary studies identified that TROY forms a novel signaling complex with EGFR and modulates EGFR survival signaling. TROY expression significantly increases the capacity of EGF to stimulate glioblastoma cell invasion while knockdown of TROY expression by shRNA or by the repurposed small molecule propentofylline blocked EGF stimulation of glioma cell migration suggesting that the TROY-EGFR complex represents an unappreciated therapeutic target to inhibit glioma invasion and decrease therapeutic resistance. Although the mechanistic basis of signaling from this novel complex on glioblastoma cell migration, invasion, proliferation, and resistance remain largely undefined, initial studies indicate that TROY signals through PDZ-RhoGEF, a guanine exchange factor that regulates the Rho GTPases RhoA and RhoC, linking TROY to cytoskeletal organization and cell motility. Knockdown of PDZ-RhoGEF significantly inhibits TROY and EGF stimulated invasion positioning PDZ-RhoGEF as a potential important effector of the TROY-EGFR complex. The overall objective of the current proposal is to determine the mechanistic basis of the interaction between TROY and EGFR and define the functional role of this unique signaling complex in glioblastoma cell invasion, survival, and therapeutic resistance. Our rationale in this proposal is that defining the mechanistic basis of TROY signaling in glioblastoma holds potential to identify candidate targets to decrease the therapeutic resistance of invasive glioblastoma cells. We hypothesize that the TROY-EGFR signaling complex enhances malignant glioblastoma cell invasion and survival. Aim 1 seeks to define the mechanistic basis of the interaction of TROY with EGFR, the specificity of the interaction, and determine its effect on glioblastoma cell invasion and resistance. Aim 2 will investigate the role of PDZ-RhoGEF as a critical effector for TROY-EGFR signaling. Aim 3 will assess the effect of inhibition of TROY expression and signaling on survival in glioblastoma xenografts as a component of combinatorial therapy. The results may provide new insights into overcoming the therapeutic resistance of glioblastoma and the identification of novel targets for improved clinical outcomes.